Various organisms have at least one enzyme that degrades the RNA of RNA-DNA hybrids. These ribonucleases H (RNases H), so far, fall into two classes based upon primary amino acid sequence similarity. From our studies, we know that the well-characterized Escherichia coli RNase HI has homologs in many different species including human and mouse. We know that these mammalian proteins resemble in sequence and function the RNase H1 of Saccharomyces cerevisiae by having a double-stranded RNA-binding activity in addition to the RNase H activity. To gain insight into the RNases H of higher eukaryotes, we have studied these enzymes from the first animal whose sequence has been completed (Caenorhabditis elegans). We find this organism has four RNase H1-like genes; one gene produces a protein typical of eukaryotic RNase H1 with a double-stranded RNA binding domain attached to the RNase H domain while the three remaining proteins vary in length but all contain a recognizable RNase H- related amino acid sequence. Both the standard RNase H1 and at least one of the additional proteins exhibit RNase H activity. The presence of multiple RNases H in C. elegans suggests that complex organisms require several enzymes of this type whereas lower organisms can survive well with only one RNase H1. Replication of the HIV retrovirus (the cause of AIDS) requires a functional RNase H that is similar in sequence and structure to cellular RNases H. Drugs are being developed that target RNase H of HIV to inhibit retroviral growth. If these drugs also interact with cellular enzymes, it is important to know the number of RNases H in cells as well as the consequences of inhibiting their actions. Even though drugs may effectively inhibit retroviral propagation, they might also create problems for normal cellular processes. Because these enzymes recognize and cleave RNA of RNA-DNA hybrids, we have examined recognition of such nucleic acids in vitro and in vivo. In vitro, examination of association of the HIV enzyme with various substrates reveals complex and interesting differences among the various duplexes as well as different enzymes. RNA-DNA hybrids are asymmetric and modifying one end of the duplex accents this asymmetry. The HIV enzyme is particularly sensitive to modifications at the termini of the RNA-DNA hybrid and exhibits different binding properties for RNA-DNA duplexes modified at each of the two ends. In vivo RNA-DNA duplexes occur during DNA synthesis, and we have shown in the yeast system that RNases H are important in both synthesis of new copies of DNA as well as in repair of damaged DNA. These results help us to understand the roles mechanism of action of these important proteins.